Bridging Project 4: Haloacid Dehalogenase (HAD) Superfamily

HAD超家族是一个由磷酸转移酶(磷酸变位酶、ATPase和磷酸酶)组成的大酶家族(约19,000个非冗余序列)[1]，存在于所有三个生命王国中[2-4]，并且在每个细胞内都有大量的同源物(在大肠杆菌中有28个；在鼠伤寒沙门氏菌中有35个；在铜绿假单胞菌中有31个；在结核杆菌中有30个；在蜡状芽孢杆菌中有31个；在脆弱类杆菌中有24个； 肺炎链球菌24株；酿酒酵母45株；秀丽线虫84株；拟南芥169株；莫登氏卷柏292株；人类183株)。多达80%-90%的成员是磷酸酶[5]，其中绝大多数功能未知。 大约40%的细菌代谢体由磷酸化的代谢物组成[6]。 磷酸取代基是常见的，因为它们提高了代谢物的水溶性，以及它与具有高亲和力和特异性的代谢酶结合的能力。从磷酸化的代谢物中去除磷酸基团是由磷酸酶完成的。一种特定的磷酸酶的“功能”是 由它在细胞中作为目标的磷酸化代谢物定义，即由它的“生理底物”定义。因此，HAD磷酸酶满足涉及磷酸化大分子和代谢物的细胞过程和代谢途径的需求。HAD磷酸酶功能的差异是基于底物识别元件的差异。底物识别元件与位于核心域的催化支架分开(图1A)。 形成活性位点的四个肽片段或“基序”位于一致的天冬氨酸亲核体、天冬氨酸/碱、赖氨酸/精氨酸和丝氨酸/苏氨酸磷酸结合残基以及镁^*辅因子天冬氨酸/谷氨酸结合残基(图IB)。这些残基与骨架主链元素结合，形成了稳定三角双锥过渡态/中间体的立体和电学模型。 沿着反应途径产生(图1B)[7]。HAD磷酸酶底物识别元件位于通过溶剂化连接子连接到核心域的帽结构域(如在HAD类C1和C2中，也称为类型I和类型11)中，或位于从核心域延伸的短环/螺旋片段中(如在“无心”HAD类CO中，也称为类型III)(图1A)[8]。 尽管HAD磷酸酶具有相同的催化位置并进行相同的第二部分反应，但它们能够利用第一部分反应的底物结合步骤和随后的加成-消除步骤的特定结构要求来区分生理底物和其他磷酸化物种(大分子和代谢物)。诱导FIT模型适用于大多数HAD磷酸酶，其中底物结合之后是帽结构域或环闭合。 底物离开基团和帽结构环之间良好的电子传递作用将有助于底物结合亲和力。为了有效地周转，磷酰基必须在催化部位内以正确的方向结合。如果底物离开基团太大或太小， 可能会发生非生产性绑定。因此，从催化部位延伸到活性部位入口的ac5位点区的大小、形状和电学表面可以提供对生理底物的身份的重要洞察。这用作使用虚拟筛选(通过结构核心和计算核心实现)来识别这里的生理底物的候选的基础。 通过实验活性筛选确定的底物专一性表明，典型的HAD磷酸酶具有松散的底物专一性和适度的催化效率。因此，仅靠活性筛查往往不能识别实际的生理底物。相反，它们提供了可使用序列/基因组分析核心和微生物学核心提供的工具进一步审问的候选基因。

The HAD superfamily is a large enzyme family (~19,000 nonredundant sequences) [1] of phosphotransferases (phosphomutases, ATPases and phosphatases) represented in all three kingdoms of life [2-4], and, within each cell, by a large number of homologs (28 in E. coli; 35 in Salmonella typhimurium; 31 in Pseudomonas aeruginosa; 30 in Mycobacterium tuberculosis; 31 in Bacillus cereus; 24 in Bacteroides fragilis; 24 in Streptococcus pneumoniae; 45 in Saccharomyces cerevisiae; 84 in Caenorhabditis elegans; 169 in Arabidopsis thaliana; 292 in Selaginella moeltendorffii; 183 in human). As many as 80-90% of the members are phosphatases [5], the vast majority of which have unknown functions. Approximately 40% of the bacterial metabolome is comprised of phosphorylated metabolites [6]. Phosphate substituents are common because they enhance the water solubility of the metabolite as well as its ability to bind to metabolic enzymes with high affinity and specificity. The removal of phosphate groups from phosphorylated metabolites is performed by phosphatases. The "function" of a particular phosphatase is defined by the phosphorylated metabolite that it targets in the cell, i.e., by its "physiological substrate". Thus, the HAD phosphatases meet the demands of cellular processes and metabolic pathways that involve phosphorylated macromolecules and metabolites. Divergence in HAD phosphatase funcfion is based on the divergence of the substrate-recognition elements. The substrate-recognition elements are separate from the catalytic scaffold, which is located in the core domain (Figure 1A). The four pepfide segments or "motifs" which form the active site position the consen/ed Asp nucleophile, Asp acid/base, the Lys/Arg and Ser/Thr phosphate-binding residues and the Mg^* cofactor Asp/Glu binding residues (Figure IB). These residues, in combinafion with the scaffold main-chain elements, form a steric and electrostafic mold that stabilizes the trigonal bipyramidal transifion states/intermediates produced along the reaction pathway (Figure 1B) [7]. The HAD phosphatase substrate recognition elements are located in either a cap domain (as in HAD classes Cl and C2, also known as Type I and Type 11) tethered to the core domain by a solvated linker, or in short loop/helical segments that extend from the core domain (as in the "capless" HAD class CO also known as Type III) (Figure 1A) [8]. Although HAD phosphatases possess the same catalytic site and proceed through the same second partial reaction, they are able to use the specific structural requirements of the substrate-binding step and the subsequent addition-eliminafion steps of the first partial reacfion to discriminate between the physiological substrate and other phosphorylated species (macromolecules and metabolites). The induced fit model, wherein substrate binding is followed by cap domain or loop closure, applies to most HAD phosphatases. Favorable electrostafic interaction between the substrate leaving group and the cap domain/gafing loops will contribute to the substrate-binding affinity. For efficient turnover, the phosphoryl group must be bound in the correct orientation within the catalyfic site. If the substrate-leaving group is too large or too small, nonproductive binding is likely to occur. Thus, the size, shape and electrostafic surface ofthe acfive site region that extends from the catalytic site to the active site entrance can provide significant insight into the identity of the physiological substrate. This serves as the basis for the use of virtual screening (made possible by the Structure Core and Computation Core) to identify candidates for the physiological substrate herein. Substrate specificities defined by experimental activity screens suggest that the typical HAD phosphatase has loose substrate specificity coupled with modest catalytic efficiency. Thus, acfivity screens alone often cannot idenfify the actual physiological substrate. Rather, they provide candidates that can be further interrogated using the tools provided by the Sequence/Genome Analysis Core and Microbiology Core.